Development of a novel loop-mediated isothermal amplification assay for ß-lactamase gene identification using clinical isolates of Gram-negative bacteria

Abstract

Rapid evaluation of antimicrobial susceptibility is important in the treatment of nosocomial infections by Gram-negative bacteria, which increasingly carry carbapenemases and metallo-β-lactamases. We developed loop-mediated isothermal amplification (LAMP)-based assays for four β-lactamase genes (blaKPC, blaNDM-1, blaIMP-1 group, and blaVIM). The assays were evaluated using eight reference bacterial strains (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter bereziniae) harboring six β-lactamase genes. A total of 55 Gram-negative bacterial strains, including 47 clinical P. aeruginosa isolates, fully characterized by next-generation sequencing (NGS), were used to evaluate the LAMP assays. The results were compared to those of conventional PCR. The LAMP assays were able to detect as few as 10 to 100 copies of a gene, compared to 10 to 104 copies for conventional PCR. The LAMP assay detected four β-lactamase genes with a sensitivity similar to that using purified DNA as the template in DNA-spiked urine, sputum, and blood specimens. By contrast, the sensitivity of PCR was 1- to 100-fold lower with DNA-spiked clinical specimens. Therefore, the LAMP assays were proved to be an appropriate tool for the detection of four β-lactamases.

Development of a Simple Method to Detect the Carbapenemase-Producing Genes blaNDM, blaOXA-48-like, blaIMP, blaKPC, and blaVIM Using a LAMP Method with Lateral Flow DNA Chromatography

“…However, some phenotypic tests require expensive instruments and special culturing conditions that are time-consuming and not available in all countries. Several LAMP-based methods involving real-time turbidimeter measurements have been reported for the detection of bla NDM , bla OXA-48-like , bla IMP , bla KPC , and bla VIM [28,29]. Although real-time turbidimeters offer high sensitivity, these instruments are costly.…”

Multi-resistance to carbapenems by the production of Imipenemase (IMP)-types carbapenemases in Gram-negative bacilli in Burkina Faso

“…Bacteria resistant to several types of antibiotics are sometime referred to as multi-resistant. Increased and inappropriate use of antimicrobial drugs promotes the emergence of antimicrobial resistant bacterial strains and related infections (Masoud et al, 2021;Kim et al, 2023). Some of those bacteria have become resistant to several antibiotics, including carbapenems and third-generation cephalosporins (Palacios-Baena et al, 2021;Arumugham et al, 2022).…”

A Multiplex Recombinase‐Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaKPC‐2, and blaNDM‐1 Genes in Klebsiella pneumoniae

“…Carbapenem‐resistant Klebsiella pneumoniae (CRKP) is a common pathogen of acquired infections in clinical practice, which can cause a wide range of infections in the organism, such as pneumonia, abdominal infections, bacteremia, urinary tract infections, brain abscesses, and other diseases, and severe infections can be life‐threatening [ 1 , 2 ]. CRKP accounts for 70%–90% of clinical carbapenem‐resistant Enterobacteriaceae (CRE) infections [ 3 , 4 ].…”

“…Molecular diagnostic methods such as qPCR are used to detect K. pneumoniae [ 11 ], but these methods are not sensitive enough and take a long time to detect. The development of fast isothermal amplification assay, such as recombinase polymerase amplification (RPA) [ 12 ], recombinase‐aided amplification (RAA) [ 13 , 14 , 15 ], and loop‐mediated isothermal amplification (LAMP) [ 1 , 16 ], have been increasingly applied for detecting a wide range of pathogens but still difficult to detect the targets in a multiplex and high‐throughput format [ 17 ].…”